The overall objective in this A2 grant is to determine the potential use of microRNAs (miRNAs) miR-15a/16 as therapy for chronic lymphocytic leukemia (CLL). This will be studied in vitro and in vivo in the murine model, NZB and in ex vivo CLL patient samples. We have described the NZB strain as a valid model of CLL that faithfully replicates the human disease. As they age, NZB develop a malignant expansion of B -1 cells with dull CD5 expression, CD11b, and B220. These mice also characteristically make an IgM anti- erythrocyte autoantibody response and polyclonal autoantibodies. We have found linkage of NZB lymphoproliferative disease with a region of mouse chromosome 14, in which there is a germline mutation in the mir-15a/16-1 loci in the 3' region flanking mir-16-1 and decreased expression of the mature miR-15a and miR-16 in NZB lymphoid sources relative to normal strain sources. A similar mutation has been found in the rare CLL patient, with the majority of CLL patients expressing deletions in this loci in the malignant clone resulting in decreased expression of miR-15a and miR-16. In the presence of decreased miR-15a/16, mRNA targets of these microRNAs are elevated including anti-apoptosis genes (bcl family) and cell cycle regulators (cyclin D1), which have been found to be increased in NZB malignant cells and critical to survival pathways for the malignant cells. An inability to respond to growth regulation and apoptosis induction may result in treatment resistant disease, much of which may be mediated by a defect in this important miRNA. The hypothesis to be tested is that abnormalities in miRNAs miR-15a and miR-16 are an underlying defect giving rise to CLL. In this A2 grant, (Specific Aim #1) normal mice will be engineered to have the NZB mir- 15a/16-1 loci to determine if the mutation results in decreased mature miR-15a and miR-16 levels, as well as effects on B-1 cell expansions. Next (Specific Aim #2), the potential therapeutic effect of in vivo lentiviral delivery of exogenous miR-15a/16 in the NZB model of CLL will be determined. The mechanism of action of miR-16 will be determined in both NZB malignant B cells (Specific Aim #3) and CLL patient samples (Specific Aim #4). The overall outcome of these studies will be to determine the potential efficacy of miR- 15a/16 delivery as a therapy or an adjuvant to chemotherapy in CLL.